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251 Mbuthia et al.
Int. J. Biosci. 2019
RESEARCH PAPER OPEN ACCESS
Interactive effects of arbuscular mycorrhiza fungi Glomus
intraradices and Trichoderma harzianum against Fusarium wilt
Lilian Wanjiru Mbuthia1, Leonard Muriithi Kiirika2*, Gloria Afolayan3, Von Alten
1Institute of Horticultural Production Systems, Section Phytomedicine, Leibniz University
Hannover, Hannover, 30419, Germany
2Department of Horticulture and Food Security, Jomo Kenyatta University of Agriculture and
Technology. P.O. Box 62000 00200 Nairobi, Kenya
3National Center for Genetic Resources and Biotechnology (NACGRAB), Moor Plantation, PMB
5382, Oyo State, Ibadan, Nigeria
Key words: Arbuscular mycorrhiza fungi, Biological control, Trichoderma harzianum, Fusarium oxysporum f.
sp. lycopersici, Synergistic.
http://dx.doi.org/10.12692/ijb/15.1.251-268 Article published on July 18, 2019
Biological control agents (BCA) are important as some establish symbiosis with plants hence controlling plant diseases,
improving plant nutrients uptake and water absorption. Use of BCA in soil borne disease management is not fully harnessed
and is also faced with inconsistencies in developing their formulations. We therefore investigated the use of arbuscular
mycorrhiza fungus (AMF), Glomus intraradices, and Trichoderma harzianum (T-22) against soilborne pathogen Fusarium
oxysporum f. sp. Lycopersici (Fol) in tomato. G. intraradices isolate 510 held on expanded clay as carrier material was
incorporated into the substrate during germination of tomato seeds and at the transplanting stage. T-22 inoculum was also
initiated from potato dextrose agar and inoculated at each transplanting stage, while Fol was applied through drenching. To
test the possible synergistic effects, AMF and T-22 were applied in combination under varying niches. Results showed that
application of AMF and T-22 together had significant reduction (30.5% p
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Int. J. Biosci. 2019
Fusarium oxysporum is an ubiquitous soilborne
pathogen that includes various pathogenic strains
which lead to economic losses in tomatoes
(Solananum lycopersicum) (Gordon and Martyn,
1997; Fravel et al., 2003; Di Pietro et al., 2003).
Chemicals applied to control Fol have been
unsuccessful as they barely target their ecological
niches but also pose dangers to the environment
(Lucas, 2006). Abuscular mycorrhiza fungi is shown
to present bio-protection effects against fungal
phytopathogens including Phytophthora, Fusarium,
Pythium and Rhizoctonia (Pozo et al., 1996; Cordier
et al., 1998; Akköprü and Demir, 2005). Its
ubiquitous nature is a fundamental component for
development, exploitation and adaptation to
interactions with the other rhizosphere microfloral of
which knowledge of mode of interactions can be
harnessed for development of an effective biological
control strategy (Linderman, 1994; Fillion et al.,
1999; Vazquez et al., 2000). Trichoderma is a
soilborne fungus commonly used as an antagonist
against fungal pathogens and as a bio-pesticide,
which is exploited for its biological control potential
(Harman, 2000; Harman et al., 2004). Moreover,
control may be achieved indirectly by inducing host
plant resistance where not only microbial organisms
have been used but also compounds that mimic their
cellular components to induce resistance (Kiirika et
The efficacy of these biological control agents and
consistence performance in controlling soil borne
diseases are the hurdles that must be overcome if
their application is to be widely used even for
commercial purposes. Attempts to combine
alternative treatments to improve performance have
been made but their application is faced with similar
constraints as the level of protection is influenced by
factors such as the pathogen strain, environment, soil
type and nutrients (Guetsky et al., 2001; Larkin and
Fravel, 2002; Spadaro and Gullino, 2005). Optimal
plant nutrition is essential for a successful disease
management where manipulation of mineral
nutrients such as nitrogen, phosphorous, sulphur and
potassium have been shown to contribute to control
of various plant diseases. Phosphorus (taken up by
plants as H2PO4– and HPO42-) is shown to have
multiple roles not only as an essential plant nutrient
but also mitigates disease development in plants
(Sweeney et al., 2000). High levels of phosphorous in
the soil however shown to reduce host’s ability to
develop AMF symbiosis (Balzergue et al., 2013). In
the current study, we investigated the interactions of
AMF and T-22 under varying phosphorous regimes
aimed at controlling Fol. Tests to determine control
effects of AMF and T-22 against Fol, tests were
carried out under different substrates containing
different P regimes. We hypothesized that application
of AMF and T-22 inhibits Fol infection and the effects
will be synergistic for combined applications.
Materials and methods
Plant materials and culture conditions
Tomato seeds of cultivar Cuor Di Bue were raised in
trays containing quartz sand and transplanted after
10 days into 9 cm diameter plastic pots (volume 230
ml). Six weeks later, seedlings were transplanted into
bigger pots of 17cm diameter (volume 2 l) and kept
under greenhouse conditions (20 oC day/night, 12 h
light per day/30 K Lux and 70% RH) and watered
regularly to the soil field capacity.
Microbial culture and inoculation
AMF and T-22 isolates were taken from the collection
of the Institute of Horticultural Production Systems,
section Phytomedicine. The inoculum of G.
intraradices isolate 510 on expanded clay as carrier
material (Dehne and Backhaus, 1986) was
incorporated into the substrate during germination
and at each transplanting stage at 5 % (v/v). T-22
inoculum was initiated from potato dextrose agar,
cultured into liquid conical flasks with sterile media
consisting of perlite pre-soaked with 2 % malt extract
solution (Kraftnahrung rein, from Villa Natura), at
2 % (v/v) into the culture substrate at each
transplanting stage. Fol inoculation was done through
drenching by pouring direct into the pot at 30 ml
spore suspension (1.5 x 108 spore/ml) at the second
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Int. J. Biosci. 2019
Substrate and fertilization
Pure quartz sand with and without 30 % (v/v) fine
ground white peat from Lithuania (calcium carbonate
at 500 g/100 l added to increase pH) were used as
substrates. Fertilization was carried out in two
nutrient regimes; Hewitt (Hewitt, 1966) low
phosphorous (LP), by 12.85 g/l NAH2PO7.H2O) and
Hewitt high phosphorous (HP) (128.5 g/l
Interactions between AMF and T-22 and their effect
when applied together on Fol were tested using
tomato cultivar Cuor Di Bue. Experiments were
conducted under greenhouse conditions, 16 h light
per day with additional light using Phillips lamps SRG
102/400 (195 µmol sec-1 m-2) during winter. Disease
progress was monitored on tomato plants growing
with temperature maintained at 22 °C (day) and 18 °C
(night) (Fig. 1). The experiment consisted of
treatments arranged in factorial design with Fol
infected or not infected (control) and subjected to the
two P-levels and substrates: (i) without T-22 or AMF
(control), (ii) with AMF alone, (iii) with both T-22
and AMF, (iv) with T-22 alone. Two conditions were
compared, during summer and winter periods which
enabled identification of differences due to changes in
different environmental conditions of the two
seasons. Furthermore, we investigated the influence
of P on Fol disease symptom expression. To observe
whether plants would start showing yellowing
symptoms of Fusarium wilt when supplied with more
P, plants with LP for AMF and control but only in the
substrate sand were included in the setup with the
aim of increasing the level of P in this extra set of
plants from LP to HP after the initial wilt symptoms
Assessments and data collection
To assess the effects of different treatments,
parameters including plant growth, disease
development, severity and incidence were considered.
AMF and T-22 establishment and intensity were
observed at different plant growth stages to monitor
their development and possible interacting effects.
Analysis of P content in the plant tissue was carried
based on the phosphorous acid colorimetric
determination method with ammonium molybdate
vanadate by Gericke and Kumries (1952) to decipher
differences in its content based on the P nutrition
level. In order to monitor disease development and
severity, visual symptoms were assessed. Symptoms
of Fol infection were observed including wilting and
yellowing and each scored separately using a scale of
0-4 according to the following classes: 0=no
symptoms, 1=< 25 % leaves with symptoms, 2=26-
50 % leaves with symptoms, 3=51-75% leaves with
symptoms and 4= 76-100 % leaves with symptoms.
Confirmation of disease incidence and development
in the plants were carried out according to
Grunewaldt-Stöcker (1994): Fol was re-isolated from
the stem cross sections at different heights (stem
base, 25 cm and 50 cm). The outer surface of the stem
was sterilised with 70 % ethanol and thin cross
sections cultured on Komada Agar (Komada, 1975).
The growth of the mycelia from the vascular bundles
was then assessed with 1/3, 2/3, 3/3 reflecting the
number of infected bundles. Re-isolation of Fol from
the substrate was done using Komada agar following
the basic dilution-plating technique: 15g of the
substrate was diluted in 150 ml sterile water, stirred
for 10min (750 rpm), a defined volume of a 10-3
dilution was then plated out on Komada agar in 3
replicates, and then incubated for 5-6 days at 23 °C
after which colonies formed per plate were counted.
This was only done for the AMF/T-22 sand HP
treatment for the first experiment as majority of
plants in the treatment showed no symptom
development and was therefore necessary to confirm
the success of inoculation by determining the
pathogen presence in the rhizosphere.
Trichoderma infection and intensity estimation
T-22 was estimated from the rhizosphere dilution-
plating at the second transplanting and harvest
stages. Briefly, quantitative isolation of T-22 was done
using selective culture medium (Steinmetz, 1994 -
unpublished): 15g of the substrate diluted in 150 ml
sterile water, stirred for 10 minutes (750 rpm) and a
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Int. J. Biosci. 2019
defined volume of a 10-3 dilution was then plated out
on TSA agar (Lewis and Papavizas, 1984) in
triplicates. The culture was then incubated for 5-6
days at 23 °C and colonies formed per plate counted
as colony forming units (CFU)/g substrate using the
AMF colonisation assay
AMF colonisation was quantified also at the second
transplanting and at harvest stages. Samples for the
assay were collected from washed root systems by
cutting a transverse band of about 1cm width at
around 5cm below the root crown. Staining was done
following Vierheilig et al. (1998): roots were soaked in
3 % KOH overnight, washed several times with tap
water, soaked overnight in ink-vinegar-solution (1 %
ink and 5 % household vinegar in a dest.). Estimation
of AMF colonisation was then done under a light
microscope (Axiolab, Zeiss) using a scale from
Backhaus (1984) as follows: 0 = no infection, 1-up to
30 % of the root section colonized, 2-up to 60 % of the
root section colonized and 3-colonisation of the whole
The statistical analysis was done using the R software,
version 2.9.0. To determine the interactions with
regard to the effect of phosphorous, substrate, and
biological control agents, a fit of an ordered logistic
regression model was used.
The model worked at estimating the probability of an
individual to get a score of 0, < 1, < 2, or < 3 for the
AMF establishment. Parameter estimates were the log
difference of odds, comparing the factors substrate,
phosphorous and the treatments. The confidence
intervals (CI) at 95 % were then calculated for the
parameters and a significant difference was shown if
the value 1 was not included in the interval. A Chi-
square test of independence with multiplicity
adjustment by Bonferroni was then done at a p-value
≤ 0.05. The model was also used to plot a model
demonstrating the differences in symptom
development in terms of wilting and yellowing as
influenced by phosphorous level at the probabilities
of getting the different scores of 1, 2, 3 or 4 and is
shown in the results segment at the probability of
getting a score of 3. For the analysis of disease
severity and Trichoderma establishment, the
treatment effects were tested using analysis of
variance (ANOVA). The significant differences were
then tested by Tukey multiple range tests (p-value
≤0.05). For disease severity, the area under disease
progress curve (AUDPC) was calculated using the
formula below and data subjected to ANOVA.
Where, xi and xi-1 are the wilt index, and ti and ti-1 are
consecutive evaluation dates (with ti-ti-1 is equal to 1
Establishment of AMF and T-22 under different
substrates and P-Levels
Analysis of interacting effects of AMF and T-22 as
well as the influence of different substrates (sand and
sand-peat mixture) and P-levels (low P and high P)
revealed that P level had a significant influence on
mycorrhiza colonisation with the logistic model
showing differences regardless of substrate, with or
Fig. 1. Tomato plants under greenhouse conditions
showing Fol wilting symptoms. Plants inoculated with
Fol (right) healthy plants without Fol inoculation
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This effect of P on mycorrhiza colonization was
evident both at the initial establishment (after 6
weeks) with a confidence interval (CI) of 3.38-16.62
and at harvest (after 13 weeks) CI of 10.02 - 23.28. A
Chi-square test of independence revealed high
significant differences in mycorrhiza colonization for
treatments under LP compared to treatments under
HP (Fig. 2A and B).
Fig. 2. AMF colonisation is influenced by different P levels (low and high levels). The graph shows data of mean
frequency values ± SD; n= 8; after inoculation with AMF alone and in combination with Trichoderma (AMF/T-
22) in the different substrates sand (SAND) and sand peat (S.PEAT) after (A) six weeks and (B) 13 weeks.
Significant differences between LP and HP in each treatment are indicated by (*) according to Chi-square test of
independence (p-value ≤0.05).
The substrate type also had significant effects on AMF
colonization with plants grown in sand showing
higher colonisation compared to sand peat at 6 weeks
(Fig. 3A and B) CI (1.18 -6.41) and after 13 weeks (Fig.
3C and D) CI (1.83-4.43). At 6 weeks, these
differences were clearly seen in the AMF/T-22 in low
P having significantly higher mycorrhiza colonisation
under sand substrate compared to sand peat (Fig.
3A). At 13 weeks, high influence was under the AMF
treatment in HP having significantly higher
colonisation in sand compared to sand peat (Fig. 3D).
AMF colonisation appeared not to be affected by the
addition of T-22 which is evidenced by almost similar
AMF-colonisation pattern levels at both the combined
treatment AMF/T-22 and individual AMF treatment
(Fig. 4A-D). The only significant difference was
detected at 6 weeks (Fig. 4B) whereby the combined
application of AMF/T-22 shows higher colonisation
in the substrate sand at LP-level.
Substrate was observed to have significant effect (p <
0.05) on T-22 establishment (Fig. 5). Treatments
under sand-peat had significantly higher levels of T-
22 compared to treatments under sand regardless of
P levels both at 6 weeks (Fig. 5A and B) and at harvest
(Fig. 5C and D). P level and AMF did not show
significant effect on T-22 establishment.
Effects of AMF and T-22 on Fusarium wilt
development under different substrates and P-levels
To determine the biological control effects of AMF
and T-22 on Fol when either applied alone or in
combination (AMF/T-22), a mixed model ANOVA
analysis of the AUDPCs under all substrate and P
levels was done. Results by ANOVA revealed
significant effect of substrate with treatments under
sand-peat which had significantly higher disease
severity compared to treatments under sand
regardless of P-level or biological control treatment
(Fig. 6A and B).
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Fig. 3. AMF colonisation is influenced by substrate sand peat and sand within the same P levels. The graphs
shows data of mean frequency values ± SD; n= 5 and 8 at (A, B) 6 weeks and (C, D) 13 weeks respectively; after
inoculation with AMF alone and in combination with T-22 (AMF/T-22) at equal levels of phosphorous.
Significant difference between the substrates Sand peat (S.Peat) and Sand in each treatment are indicated by (*)
according to Chi-square test of independence (p-value ≤0.05).
Results are also presented as disease progress curves
for the treatments with T-22 and AMF at LP or HP
both during summer and winter periods (Fig. 8)
where the developmental trend was similar for all the
A Tukey’s multiple range tests was then done to
compare treatments within the same substrate and
under the same P-levels (Fig. 8A-H) While no
significant differences were detected in the substrate
sand peat at either HP or LP, the disease progress
curves show the combined treatment of AMF/T-22
having the lowest severity level compared to the
control (Supplementary fig. 1A and B) The most
pronounced and significant effect was observed in the
substrate sand with HP where the combined
treatment AMF/T-22 showed a significant lower
disease severity compared to all the other treatments
(Fig. 1C) Re-isolation of the pathogen from the
substrate of this particular treatment confirmed
pathogen presence in the treatment (data not shown).
In the sand substrate at LP, no significant differences
were detected between the treatments with no clear
trend in the disease progress curves, but in the AMF
treatment, the trend was lower compared to others
(Fig.8D). The re-isolation of the pathogen incidence
from the stems from all the treatments inoculated
with Fol was consistent with the visual wilt
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development scores (data not shown) with the plants
showing symptoms visually also showing infection in
the selective agar media, while those that did not have
any symptoms visually not showing any infection.
The second experiment was done in winter and
allowed us to observe the differences due to different
environmental conditions as compared to those of
summer in the first experiment. The greenhouse
temperature during summer period ranged from 28-
32 °C while in winter, temperature was maintained
between 22-25 °C. Establishment of AMF and T-22
was again determined at initial stages and at harvest
but this was only done to confirm their establishment.
For T-22, this was done in both treatments containing
T-22 and in the control to determine whether there
was any indigenous Trichoderma spp. in the
substrates used. The presence of T-22 in the
rhizosphere of treatments inoculated with T-22 was
confirmed, but no indigenous T-22 was detected in
the controls. The colonisation of AMF on the roots
treated with AMF was also confirmed.
Fig. 4. AMF colonisation is influenced by addition of T-22. The graphs shows data of mean frequency values ±
SD; n= 5 and 8 at (A, B) 6 weeks and (C, D) 13 weeks respectively; after inoculation with arbuscular mycorrhiza
fungi (AMF) alone and in combination with Trichoderma (AMF/T-22) at the same phosphorous levels within the
same substrate, Sand peat and Sand. Significant difference between AMF and AMF/T-22 at the same
Phosphorous level (LP or HP) is indicated by (*) according to Chi-square test of independence (p-value ≤ 0.05).
In general, the disease incidence and severity levels in
winter were lower than in summer and so was the
progress of disease development which took four
weeks in winter as compared to only two weeks in
summer. Due to the fact that the incidence and
severity levels were very low in winter (Fig. 8E-H)
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Int. J. Biosci. 2019
with only half of the plants within each treatment
showing symptoms, no statistical analysis was
possible and therefore only the trends of the disease
progress curves are compared. The disease progress
curves indicate that the biological control agents AMF
and T-22 alone or combined also reduced the severity
levels of the disease in winter as their progress curves
are lower in comparison to the controls. The other
trends on the effect of P and substrate on disease
development was the same as in the first experiment.
Fig. 5. Colony forming units (CFU) of T-22 as influenced by substrates, P and AMF. Included are the ± standard
deviation; n= 5 and 8 at (A, B) 6 weeks and (C, D) 13 weeks respectively, after inoculation with T-22 alone or in
combination with AMF (AMF/T-22) under the same substrate Sand or Sand peat and P levels ( LP or HP) at 6
weeks and at 13 weeks.
Effect of P on Fusarium wilt development
From our investigations, we observed an unexpected
influence of P on fusarium wilt symptom
development. It was interesting to note that plants
grown under low P did not develop the typical
expected symptoms associated with Fusarium wilt. A
prominent and distinctive symptom development was
observed between plants under HP and LP. Plants
under HP developed typical Fol symptoms of first
yellowing then gradually wilting while those under
low P directly wilted without yellowing.
This difference was clear across the whole data set
regardless of the treatment or substrate. To
demonstrate this, the scores of both the wilting and
yellowing symptoms were fit into a logistic regression
model showing the probability to get a score of 1, 2, 3
or 4 within the period of disease development.
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Fig. 6. Disease severity is influenced by substrate within the same P levels. The figure shows disease progress
curves within 18 days for treatments with T-22, at (A) low phosphorous level ; (B) high phosphorous level.
The model was then able to show the disease progress
curves of both wilting and yellowing and how this was
influenced by the P level across the whole data set.
This is best shown and illustrated here at the
probability of getting a score of 3 (Fig. 9) and was
clearly observed on plants (Fig. 10).
Fig. 7. The P concentration in plant tissue in treatments with high HP and LP level. Included are the ± standard
deviation; n= 10 in treatments with AMF and without any biological control (control) under the same substrate
sand or sand peat (S.Peat). Significant differences in HP and LP within the same treatment are indicated by (*)
according to Tukey multiple range test (p-value ≤ 0.05).
Plant tissue analysis of control and AMF treatments
was done to determine actual differences in P content
for the treatments with HP and LP. The ANOVA
showed interaction effects at P and substrate with no
interactions at the treatment levels. This is shown in
(Fig. 7) where HP has significant higher
concentration levels than LP. The substrate
interaction effect was brought out by a significant
difference between the treatments in sand having
higher concentrations than those in the sand peat at
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Fig. 8. The effect of AMF and T-22 inoculations on disease severity in summer and winter periods. The figure
shows disease progress curves for treatments with AMF, T-22, their combination AMF/T-22 and without AMF or
T-22 (control) as they occurred in summer (A, B, C, D) and winter (E, F, G, H) in treatments within the same
substrate and P-levels.
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To further ascertain the effect of P on Fusarium wilt
symptom development and expression, a sub-
experiment was set-up. A sub-set of treatments (AMF
and control under substrate sand) were first grown
under LP levels, inoculated with Fol and upon
symptom development, the P level was then increased
to the HP level. The idea was to increase the level of P
to that of HP after the initial wilt symptoms were
detected and observe whether these plants would
start showing the yellowing symptoms with the P
increase. The disease development period was not
long enough i.e. 18 days from initial symptom
development to maximum severity level to clearly
show the effect of increasing the P level. Within that
period, fertilization with the increased level of P had
been done only two times. Nevertheless, the
observations made towards the end of the experiment
showed that, after increasing the level of P, plants
began to wilt and started showing yellowing
Synergistic effects of AMF and T-22 by dual
inoculation against Fusarium wilt of tomato, with the
aim to contribute to development of effective
biological control strategies against soil-borne
pathogens. Preliminary investigations on the
influence of the two biological control agents on each
other based on establishment were conducted and no
antagonistic effects were observed. Establishment
levels of T-22 and AMF were similar in the dual
inoculation in comparison to their individual
inoculation implying that both organisms have a non-
Fig. 7. A logistic regression model showing the distinct disease symptom expression (wilting and yellowing) as
influenced by P levels. Different treatments starting from top to bottom which include; (a) T-22, (b) Control, (c)
AMF/T-22 and (d) AMF. The columns indicates the P level and substrate starting from the left with; Sand Peat
HP, Sand Peat LP, Sand HP and Sand LP. The straight line and the dotted lines represent the yellowing and
wilting curves, respectively.
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The trend of wilt symptom development shows that
the combined treatments had lower severity levels
than the individual treatments as well as the control.
This may be attributed to synergistic or additive
effects between AMF and T-22. Synergy between
mycorrhiza and Trichoderma spp. on enhancement of
growth and biological control effects were reported
earlier (Camprubi et al., 1995; Datnoff et al., 1995;
Fillion et al., 1999).
The most significant and conspicuous observation
was observed with sand the sand substrate with HP
whereby the combined treatment of AMF/T-22
showed significantly minimal disease infection
compared to the single inoculated treatments or the
control. Re-isolation of the pathogen from the
substrate of this particular treatment confirmed its
presence, while the analysis of the pathogen incidence
from the stems gained no growth on the selective agar
media. This indicates mechanisms other than
competition and mycoparasitism. Thus, a possibility
of synergistic induction of resistance in the dual
application could have played a key role which
remains to be investigated. Cross-talk between AMF
and Trichoderma during interactions with the host
plant affects disease resistance through their
influence on phytohormone synthensis or transport.
Martinez-Medina et al. (2011) reported that the co-
inoculation of AMF and Trichoderma in Cucumis
melo increased the hormonal profile of SA and JA
that plays a key role during plant resistance responses
as compared when applied singly. Thus, from our
observations, the AMF colonisation took place
congruent to pathogen attack which combined with
the presence of T-22 resulted in the minimal disease
infection observed in the AMF/T-22 sand HP
treatment. However, the results give no evidence that
mycorrhiza was essentially responsible for that effect,
as there was no difference in AMF colonisation
compared to sand peat treatment. Also, the presence
of T-22 in lower density in sand than in the sand peat
also gives no real explanation for the increased
disease suppression. Consequently, it would only
suffice to conclude that other components and/or
factors of that rhizosphere condition may have
enhanced or complimented the defence mechanisms,
the speculations that remain to be investigated.
The enhancement of the resistance levels caused by
both AMF and T-22 towards attainment of high
resistance has been investigated using Trichoderma,
where transformants with over-expression of
chitinase genes were shown to confer higher defence
(Howell, 2003). Improved biological control of Fol by
AMF has also been achieved by the combination of
AMF with hormonal elicitors like salicylic acid and
jasmonic acid (El-Khallal, 2007). On the other hand,
research on the effect of soluble substances released
by extra-radical mycelium of AMF (Fillion et al.,
1999) showed evidence that these substances have
some stimulatory effect on the growth of
Trichoderma. So, the question would be whether
there is also a possibility that AMF may enhance the
production of lytic enzymes in Trichoderma which
can be studied via molecular techniques.
Furthermore, the speculative influence of T-22 and
AMF on each other require also further investigations
either by: a) comparing the dual application of
AMF/T-22 to that of a Trichoderma transformant e.g.
without chitinase with the assumption that it`s
production would be the prominent mechanism of
action leading to the highly induced mechanism in
the dual application or, b) Comparing the resistance
conferred by application of AMF/T-22 to that of
AMF/JA-SA or to that of a transgenic resistant variety
by monitoring the gene expression.
Our results showed that application of P influences
Fol disease development as manifested by reduced
symptom expression. This could not only be
attributed to the function of P as an essential mineral
element required by plants but also its indirect role in
mitigating disease development. Thus, healthy and
vigorous root system will better compensate for
infections especially by roots pathogens. Plants under
low P application exhibited higher disease severity
and wilted directly without the typical Fol symptom
development process involving yellowing of leaves as
observed under high P. P treatment was shown to
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Int. J. Biosci. 2019
influence the population of Fusarium spp. by affecting
the structural population density, virulence and
disease incidence levels (Woltz and Jones, 1973;
Yergeau et al., 2006). Yergeau et al. (2006) reported
the effects of varying P regimes on Fusarium crown
and root rot of Asparagus officinalis, where disease
incidence was significantly low for plants under low P.
Direct wilting of plant under low P regime further
shows the essential role of P during the plant’s
metabolic processes. As an essential mineral element
that functions as plant structural compound, P is
shown also to act as a catalyst in various biochemical
reactions in plants (Marschner, 1995; Raghothama,
1999; Raghothama, 2000; Walters and Bingham,
2007). Under optimal supply, P is taken up in the
form of orthophosphate (Pi) and translocated into the
cytoplasm (Jain et al., 2005). However, under low P
supply in the soil, the intracellular Pi can be reduced
significantly in the cell affecting the supply of key
energy carrier molecules such as ATP/ADP that are
essential for plant cellular biochemical processes such
as photosynthesis (Marschner, 1995; Rao, 1997;
Raghothama, 1999; Jain et al., 2005). Low Pi
concentration in the cytoplasm affects photosynthetic
process by suppressing the expression of light-
regulated psbO and psbP genes that encode oxygen-
evolving proteins of PSII complex (Jain et al., 2005).
Thus, due to the suppressive effects of P deficiency in
the cells, physiological adaptive changes that
culminate to hampered photosynthesis occurred
leading to direct wilting without yellowing.
Fol infection was shown to limit the photosynthetic
rates of tomato leaves by decreasing the light-
saturated rate of CO2 assimilation accompanied by
decreased maximum carboxylation velocity and in
turn reducing the maximum quantum efficiency of PS
II (Nogues et al., 2002).
Results on plant tissue analysis showed that the
concentration of P was significantly reduced in plants
grown under low P as compared to high P infected
with Fol. Pi is critical for energy metabolism in the
host plant which implies that processes such as
photosynthetic activity were interfered with.
Yellowing of leaves is usually associated with
chlorophyll breakdown, which is an elaborate and
highly energy consuming process (Matile et al., 1996).
Since plants with low P are expected to have a lower
metabolic energy due to the significantly reduced
photosynthesis, this process would cause a
physiological blocking of the chlorophyll breakdown
in a bid to save energy, resulting in expression of
Fusarium symptoms only by wilting.
The role of P was further shown by replenishing the
LP plants with additional P in order to attain the HP
status but after the onset of initial symptoms.
Interestingly, a similar process of direct wilting as for
the plants under HP was observed where plants first
showed the yellowing symptoms (data not shown).
Thus, P application delayed wilting symptoms as
observed also with AMF treatment. Previous studies
showed that the effects of low Pi on photosynthesis
can be reversed by increasing the levels of P to the
optimum (Rao, 1997; Jain et al., 2005). Moreover,
Jain et al., (2005) observed that increased P content
lead to the up-regulation of the genes associated with
photosynthesis that had initially been down regulated
due to P deficiency. Precise mechanisms underlying
the effects of P on Fusarium symptom expression due
to P deficiency is not clearly understood. Molecular
and proteomic tools could possibly be used in future
studies to understand the cellular metabolic activities
ensued upon Fusarium infection, where potential
genes or protein components that most likely support
the possible physiological adaptive mechanisms could
Results of our investigations shows that AMF and T-
22 can co-exist and interact with each other in the
rhizosphere without having any detrimental or
antagonistic effects on their establishment.
Furthermore, we report also the synergistic effects of
AMF and T-22 against the soil-borne pathogen Fol
which contribute to development of soil borne disease
management strategies. Application of AMF in
combination with other biological control organisms
can further be studied to reduce their variability in
264 Mbuthia et al.
Int. J. Biosci. 2019
efficacy and determine the appropriate application
This has also been shown in several other studies with
AMF, whose effects are shown to be enhanced either
by combining with biological control organisms or
elicitors like salicylic acid and jasmonic acid.
However, interacting factors that affect and
manipulate their efficacy remain to be elucidated in
order to exploit the full potential of the biological
control organisms with the keen interest of
conserving the environment by reducing pesticide use
in cropping systems. Our study shows that substrates,
P nutrition as well as environmental conditions
influences the establishment, growth and
development of the biological control agents used as
well as that of the pathogen.
These findings can be used in predicting how these
biological control agents would develop under certain
conditions that could be used to understand the
functioning of different biological systems, hence
facilitating development of highly specific and reliable
biological control strategies.
This research was supported by the Institute of
Horticultural Production Systems, Section
Phytomedicine, Leibniz University Hannover,
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